The process combining supercritical CO2 and co-sterilants produces strong synergistic microbicidal effects in relatively mild conditions. The mechanism of microbe inactivation is not well characterized, but the disruption of lipid membranes and mild oxidative damage appear to be involved. The process, which is also highly efficient against viruses, is considered to be very mild compared to other sterilization methods. Experimental evidence shows that damage to protein is limited, and that immunogenic epitopes can survive the process. In this project, NovaSterilis scientists and the group of Bryce Chackerian at the University of New Mexico will use both bacteriophages and recombinant Virus-Like Particle vaccines to optimize this sterilization process to obtain high virus inactivatin rates in combination with limited protein damage and strong epitope integrity. The process optimization will include varying sterilant concentrations, and exposure time, temperature and pressure in the supercritical environment. Viral and VLP particle integrity will be assessed by electron microscopy, gel electrophoresis and size exclusion chromatography. Protein damage analysis will include fragmentation, aggregation and carbonylation assays. Epitope integrity will be assessed by ELISA. Future development will further characterize the process with different types of animal viruses, study damage to proteins, nucleic acids and lipids, and demonstrate in vivo that scCO2-sterilized VLP vaccines still induce a strong immune response and protection against pathogen challenge. The developed process will be applicable to the production of inactivated vaccines, and the final sterilization of labile biopharmaceuticals and sub-unit vaccines. A better understanding of methods mitigating protein damage in the sterilization process will have broader applications, including protecting enzymes and growth factors in supercritical-based tissue engineering.